KR100807643B1 - Diluent for boar semen and liquid boar semen using the diluent - Google Patents

Abstract

A boar semen diluent for artificial insemination is provided to be simple for preparation, be able to be stored in a general refrigerator at a temperature of 4 deg.C, and be used for 5 days at any time, thereby being easy for artificial insemination in swine farming and in vitro fertilization in laboratories and significantly contributing to species improvement of the swine and the study of a biological engineering field. A boar semen diluent for artificial insemination comprises 30-40 grams of glucose, 10-15 grams of TES(N-tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid), 1-3 grams of Tris[hydroxymethyl]aminomethane, 0.1-2 grams of KCl, 0.1-2 grams of N-acetyl-D glucosamine, 150-250 ml of yolk, and 2-5 grams of BSA(Bovine serum albumin) per 1,000 ml of an aqueous solution.

Description

Diluent for boar semen and liquid boar semen using the diluent}

The present invention relates to artificial insemination diluent of pig sperm liquid and pig liquid sperm using the same, more specifically, when preparing a pig liquid sperm using the pig sperm diluent according to the present invention, even if stored at 4 ℃ for 5 days It relates to a dilute solution of artificial insemination pigs excellent in motility, tricuspid ratio and viability of the pigs and liquid liquid pigs using the same.

Artificial insemination can increase the productivity of pig farms by increasing the use efficiency of high quality sows compared with natural seedlings, but many pig farmers have avoided artificial insemination due to the stereotype of low conception rate and low live birth. In recent years, however, the need for artificial insemination is increasing due to the expansion of pig breeding and the cooperative or complexation of breeding forms, and most pig farmers are carrying out artificial insemination.

In case of using undiluted semen without diluting the semen collected during artificial insemination, there is a restriction that artificial insemination should be performed within 2 hours after collecting semen. Therefore, in general, liquid semen or freezing by diluting artificially extracted semen It is stored in a semen state and used to correct it when necessary. Copper crystals have the advantage of storing semen for a long time, but liquid sperm is generally used because of low birth rate and low litter count. As the liquid sperm has a large difference in the delivery rate and the number of litters, depending on the storage temperature and duration, as well as the type of diluent used in the manufacture of the liquid sperm, many researches and developments on diluents that can increase the success rate of artificial insemination have been made. .

Various kinds of liquid liquid semen are used by commercial artificial insemination centers around the world. Modena (Moretti, 1981, In Proceedings of the 8th International Pig Veterinary Society Congress, Ghent, Belgium, p. 293) is one of the most commonly used dilutions. Summermatter (1984, Verterinar-Humanmedizinische Gemeinschaftstagung, Hannover, FRG, pp. 72-75) developed a Butschwiler diluent that modulated Modena, adding cysteine and bovine serum albumin (BSA) and increasing the proportion of glucose. It was. The dilutions are generally dilutions that are stored at 15-18 ° C.

Most of the diluents required for artificial insemination of pigs are imported from foreign countries, or they are dependent on the use of clones produced in Korea by receiving the diluent manufacturing technology. In addition, there is a difficulty in storage requires a diluent that can be manufactured by a simple manufacturing process and can be stored in a general refrigerator (4 ℃).

Chung et al. (1989, Korean J. Anim. Sci. 31: 58-161) reported liquid sperm that can be preserved at 5 ° C. for 9 days by injecting a 5 ml straw with a BF5 diluent. Park et al. (2004. Asian-Aust. J. Anim. Sci. 17 (10): 1369-1373) modified the lactose yolk (LEY) by adding 0.05% N-acetyl-D-glucosamine to 4 ° C. The semen dilution, LEN, was reported.

In general, a mammalian injected ejaculated semen gains the ability to invade oocytes after high concentrations of spermatozoa have been cultured for a certain period of time (capacitation). Incubation time of the injected crude solution for successful fertilization is 4-8 hours (Hamano and Toyoda, 1996, Jpn. J. Anim. Reprod. 32: 177-183; Suzuki et al. 1986, Anim. Sci.Tech.Jpn. 67: 24-27). However, sperm invasion in in vitro fertilization of frozen-thawed sperm was shown without culture time (Wang et al., 1991, J. Reprod. Fertil. 93: 491-496). Watson (1995, Reprod. Fertil. Dev. 7: 871-891) reported that freeze-thawed sperm had already begun to acquire fertility, ie the cell membranes of freeze-thawed sperm had not frozen and fertility gain had occurred. It is similar to sperm and reported that it is similar to the acrosome reaction that occurs for invasion of oocytes. In addition, fertilization gaining sperm increased during cooling from room temperature to 5 ° C., and this fertilization gaining sperm does not require incubation time after freeze-thaw for oocyte invasion. However, the mechanism of fertility gain results without culture time is unknown (Martinez et al., 1996, Reprod. Dom. Anim. 31: 317-320, 1996).

However, LEN has a problem in that the success rate of artificial insemination and in vitro fertilization is greatly reduced due to the significant decrease in sperm motility, normal acrosome ratio and viability of liquid sperm with incubation time. Therefore, a diluent capable of securing stability of the liquid sperm during the cultivation to obtain fertility as well as the storage period is required and the liquid crystalline solution thereby.

The present invention is to solve the problems of the prior art as described above, because the manufacturing process is simple and excellent in sperm motility, normal acrosome rate and sperm viability even when stored at 4 ℃ can be used at any time while storing in a normal refrigerator It is to provide a diluent of pig liquid sperm and liquid crystalline solution thereby facilitating artificial insemination in pig farms and in vitro fertilization in the laboratory.

Pigment diluent for artificial insemination of the present invention for achieving the above object is 30 ~ 40g glucose per 1,000ml aqueous solution, 10 ~ 15g TES (N-tris [Hydroxymethyl] methyl-2-aminoethane-sulfonic acid), Tris [ hydroxymethyl] aminomethane 1-3g, 0.1-2g potassium chloride (KCl), 0.1-2g N-acetyl-D-glucosamine, 150-250ml egg yolk, and bovine serum albumin (BSA) ) 2 to 5 g, characterized in that the composition.

Although the data are not specifically described in the Examples, in order to determine the content of each component in the composition, the concentration of N-acetyl-D-glucosamine in 100 ml solution was diluted, and the normal acrosome rate was examined after diluting the pig semen. The efficiency was high when 0.1 ~ 2g was added. In particular, when 0.5g was added, the normal acrosome rate was the highest.

In order to determine the range of egg yolk, the normal acrosome rate was tested by changing the yolk concentration from 0 to 25% in a solution containing N-acetyl-D-glucosamine. It was highest at and more than 20% was more effective than no addition, but the efficiency decreased again.

In the same manner as described above, glucose, TES, Tris, KCl and bovine serum albumin in the composition were added in order to determine the composition range of each component.

In the composition of the present invention glucose acts as a metabolic substrate and energy source, TES, Tris, KCl serves to regulate the buffering and acidity (pH) and osmotic pressure of sperm.

As can be seen from the embodiment, the liquid sperm prepared by the artificial insemination diluent solution for fertilization according to the present invention is superior to the conventional liquid distillation liquid sperm sperm motility, normal acrosome rate and viability, so to increase the success rate of artificial insemination Can be. Conventional liquid crystals are difficult to store, but the present invention has the simplicity that can be stored in a general refrigerator of 4 ℃ and can be used at any time for five days, its utility is large.

Hereinafter, the sperm motility, normal acrosome rate, and viability of the sperm of the conventional liquid diluent LEN and the liquid sperm using the diluent GPL4 according to the present invention were compared through the Examples.

Example

Example 1: Pigment diluent for artificial insemination (GPL4)

Using the components and contents shown in Table 1 below to prepare a diluent solution for artificial insemination pigs according to the present invention. The remaining portions other than the components listed in Table 1 were adjusted in volume using purified water. In the preparation of GPL4, the BSA may cause a change in the BSA properties due to the foaming caused by the stirring at the time of addition.

Example 2 liquid liquid GPL4

The concentrated concentrated solution at 37 ° C. collected by the hydraulic method was cooled to room temperature (20-23 ° C.) within 2 hours after collection, and then diluted to 5 × 10 ⁹ sperm / 10 ml at room temperature using the GPL4 solution prepared in Example 1. Pigment liquid was prepared. The liquid sperm prepared in each 10ml 15ml test tube was stored in a 4 ℃ refrigerator for 5 days and examined the motility, normal acrosome rate and viability of the sperm of the following example.

Comparative Example: LEN pig liquid sperm

A liquid liquid swine was prepared in the same manner as described in Example 2, except that LEN (containing 110.0 g of lactose hydrate, 0.5 g of N-acetyl-D-glucosamine and 200 ml of egg yolk in a 1,000 ml aqueous solution) was used. . The prepared liquid sperm was stored in a 4 ° C. refrigerator together with the liquid sperm of Example 2 for 5 days and the motility, normal acrosome rate and viability of the sperm of the following examples were investigated.

Example 3 Characteristics of Porcine Liquid Sperm

1) Constant Temperature Culture of Liquid Sperm

100 μl of the sample was taken every day while storing the liquid crystals of Example 2 and Comparative Example at 4 ° C. for 5 days, diluted in 2 ml of BTS (Beltsville thawing solution), and incubated at 38.5 ° C. for each time indicated in the table. Samples were taken and tested for properties.

In order to examine the characteristics of the liquid sperm prepared by using the artificial cultivation diluent for the cultivation of the present invention, the sperm motility, normal acrosome rate and viability were assayed using the liquid sperm incubated in 1) in comparison with the comparative example. It was. Each experiment was statistically repeated five times, and in all the tables below, if abcd is represented in the same row and xyz is represented in the same column in the same column, the significance is recognized at the 0.05% level between the averages. it means.

2) sperm motility test

Samples incubated in 1) were collected in 0.5, 3, and 5 hours of incubation, respectively, and recorded on a video screen, recorded on a video tape, assaying the motility (%) with a normal microscope (250x). The results are shown in Table 2 and Table 3. Table 2 shows the results for GPL4 liquid crystals, and Table 3 shows the results for LEN liquid crystals. As can be seen from the comparison between Table 2 and Table 3, the motility of sperm in the GPL4 liquid sperm according to the present invention was excellent even after storage at 4 ° C. for 5 days and showed more than 5 times higher motility than the LEN liquid sperm. In addition, the sperm motility is maintained even after the incubation time compared to the liquid LEN liquid according to the prior art is expected to significantly increase the conception rate and litter count.

3) normal acrosome rate assay

Samples incubated in 1) were taken in incubation in 0, 3 and 5 hours, respectively, fixed with 1% glutaraldehyde (glutaradehyde) and examined for normal acrosome rate with a phase contrast microscope (1,000x). And Table 5. Table 4 shows the results for GPL4 liquid crystals, and Table 5 shows the results for LEN liquid crystals. As can be seen from the comparison between Table 4 and Table 5, the normal acrosome ratio of the GPL4 liquid sperm according to the present invention was remarkably excellent in the LEN liquid sperm after storage at 4 ° C. for 5 days and after incubation.

4) Sperm Viability  black

Samples incubated in 1) were collected in incubation at 0, 3 and 5 hours, respectively, and stained with SYBR-14, which marks live sperm, and propidium iodide fluorescence staining, which examined dead sperm. Are listed in Table 6 and Table 7. Table 6 shows the results for GPL4 liquid crystals, and Table 7 shows the results for LEN liquid crystals. As can be seen in the comparison between Table 6 and Table 7, the viability of sperm in the liquid GPL4 liquid crystals according to the present invention was excellent in LEN liquid crystals after storage at 4 ℃ for 5 days and even after incubation.

As described above, the artificial insemination diluent for pig fertilization and the liquid pig insemination according to the present invention have a simple manufacturing process, have a simplicity that can be stored in a general refrigerator at 4 ° C., and can be used at any time for five days. Easy fertilization and in vitro fertilization in the laboratory can greatly contribute to breeding of pigs using excellent sows and research in biotechnology.

Claims (2)

30 ~ 40g of glucose per 1,000ml aqueous solution, 10 ~ 15g of TES (N-tris [Hydroxymethyl] methyl-2-aminoethane-sulfonic acid), 1 ~ 3g of Tris [hydroxymethyl] aminomethane, 0.1 ~ 2g of potassium chloride (KCl), N- N-acetyl-D-glucosamine (N-acetyl-D-glucosamine) 0.1 ~ 2g, egg yolk 150-250ml and bovine serum albumin (BSA, Bovine serum albumin) characterized in that it contains a diluent solution for pig fertilization . delete